Anti wnt 7b

The immunohistochemistry staining was done as described previously (Wang et al., 2011 (link); Hahn et al., 2013 (link)). Each antibody staining was
performed on multiple brain sections and the experiment was repeated one or more
times. Primary antibodies are as follows: anti-DLX2 (Kuwajima et al., 2006 (link)), anti-SATB2 (abcam, ab51502,
1:200 dilution), anti-SOX2 (Santa Cruz Biotechnology, sc-17320, 1:200 dilution),
anti-SOX1 (R&D Systems, AF3369, 1:100 dilution), anti-SOX5 (Santa Cruz
Biotechnology, sc-20091, 1:200 dilution), anti-CNTN2/TAG-1 (Developmental
Studies Hybridoma Bank, 4D7/TAG1, 1:10 dilution), anti-WNT-7b (Novus,
NBP1-59564, 1:200 dilution). Secondary antibodies with minimal cross reactivity
to other species were obtained from Jackson ImmunoResearch. Images were taken
using a confocal microscope (Zeiss LSM510 META NLO Axiovert 200M Inverted) or a
fluorescence microscope (Olympus IX81 Automated Inverted). LSM Image Browser and
Image-Pro Premier were used for image process and quantification.

Tissue was fixed in 10% formalin, paraffin embedded, cut into 4μm sections and put on slides. Deparaffinized sections underwent antigen retrieval in citrate buffer (Dako) for 30 minutes, blocked for 4 minutes in Background Sniper (Biocare Medical). Sections were incubated with primary antibodies overnight at 4°C and secondary antibodies for 2 hours at room temperature, then incubated with ABC complex (Vectastain) and VECTOR Red (Vector Laboratories) or HSS-HRP and DAB (Vector Laboratories), then stained with hematoxylin. Immunofluorescence sections were incubated with Hoechst for 45 minutes at room temperature. Antibodies: anti-PanCK (Dako #Z0622), anti-hepatocyte (Biocare Medical #166), anti-JAG1 (Santa Cruz #8303), anti-KRT19 (DSHB), anti-WNT7B (Novus Biologicals #NBP1-59564), anti-GFP (Cell Signaling #2956), biotinylated anti-rabbit (Vector BA-1000), Alexa Fluor 488 α-rabbit (Thermo A-11008), Alexa Fluor 555α-rat (Thermo A-21434).